Abstract
PurposeExtracellular vesicles (EVs) contain RNA and protein cargo reflective of the genotype and phenotype of the releasing cell of origin. Adult neural retina EV release, RNA transfer, and proteomic cargo are the focus of this study.MethodsAdult wild-type mouse retinae were cultured and released EV diameters and concentrations quantified using Nanosight. Immunogold transmission electron microscopy (TEM) was used to image EV ultrastructure and marker protein localization. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze retinal cell transcripts present in EVs. Super-resolution microscopy was used to image fluorescent (green) RNA and (red) lipid membrane labeled EVs, released by adult retina, and internalized by isolated retinal cells. Mass spectrometry was used to characterize the proteomes of adult retina and EVs.ResultsAdult neural retina released EVs at a rate of 1.42 +/− 0.08 × 108/mL over 5 days, with diameters ranging from 30 to 910 nm. The canonical EV markers CD63 and Tsg101 localized to retinal EVs. Adult retinal and neuronal mRNA species present in both retina and EVs included rhodopsin and the neuronal nuclei marker NeuN. Fluorescently labeled RNA in retinal cells was enclosed in EVs, transported to, and uptaken by co-cultured adult retinal cells. Proteomic analysis revealed 1696 protein species detected only in retinal cells, 957 species shared between retina and EVs, and 82 detected only in EVs.ConclusionsThe adult neural retina constitutively releases EVs with molecular cargo capable of intercellular transport and predicted involvement in biological processes including retinal physiology, mRNA processing, and transcription regulation within the retinal microenvironment.
Highlights
Extracellular vesicles (EVs) contain RNA and protein cargo reflective of the genotype and phenotype of the releasing cell of origin
The mammalian retina releases a range of extracellular soluble factors active in development, neurophysiology, disease and neuroprotection.[1,2,3]
Established soluble factors present during retinal development that are involved in cell division and fate determination include basic fibroblast growth factor, transforming growth factor alpha (TGFα), and leukemia inhibitory factor (LIF)
Summary
Adult wild-type mouse retinae were cultured and released EV diameters and concentrations quantified using Nanosight. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze retinal cell transcripts present in EVs. Super-resolution microscopy was used to image fluorescent (green) RNA and (red) lipid membrane labeled EVs, released by adult retina, and internalized by isolated retinal cells. The vitreous and retinal pigment epitheliums (RPE) were removed from neural retinas. Neural retinas were cultured in 6-well plates at 37°C in 5 mL of Dulbecco’s Modified Eagle Medium (DMEM), plus 10% exosome free fetal bovine serum (FBS), 1% penicillin-streptomycin (Millipore Sigma) and 0.2% Nystatin (Millipore Sigma). Retina conditioned media and retinal tissues were collected after five days for analysis
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